The present invention relates generally to model systems for diseases that involve defects in the function of mitochondria and specific mutational burdens. The invention also relates to the use of these model systems for screening drugs and evaluating the efficacy of treatments for those diseases. In particular, the invention relates to the diagnosis and treatment of late onset diabetes mellitus and related pathologies, such as impaired glucose tolerance and insulin dependent or non-insulin dependent diabetes.
Diabetes mellitus is a common degenerative disease affecting 5 to 10 percent of the population in developed countries. It is a heterogeneous disorder with a strong genetic component; monozygotic twins are highly concordant and there is a high incidence of the disease among first degree relatives of affected individuals. The propensity for developing diabetes mellitus is reportedly maternally inherited, suggesting a mitochondrial genetic involvement. (Alcolado, J. C. and Alcolado, R., Br. Med. J. 302:1178-1180 (1991); Reny, S. L., International J. Epidem. 23:886-890 (1994)).
Studies have shown that diabetes mellitus may be preceded by or associated with certain related disorders. For example, it is estimated that forty million individuals in the U.S. suffer from late onset impaired glucose tolerance (IGT). IGT patients fail to respond to glucose with increased insulin secretion. A small percentage of IGT individuals (5-10%) progress to insulin deficient non-insulin dependent diabetes (NIDDM) each year. Some of these individuals further progress to insulin dependent diabetes mellitus (IDDM). This form of NIDDM or IDDM is associated with decreased release of insulin by pancreatic beta cells and/or a decreased end-organ response to insulin. Other symptoms of diabetes mellitus and conditions that precede or are associated with diabetes mellitus include: obesity, vascular pathologies, peripheral and sensory neuropathies, blindness, and deafness.
Due to the strong genetic component of diabetes mellitus, the nuclear genome has been the main focus of the search for causative genetic mutations. However, despite intense effort, nuclear genes that segregate with diabetes mellitus are known only for rare mutations in the insulin gene, the insulin receptor gene, the adenosine deaminase gene and the glucokinase gene.
Clearly, a reliable diagnosis of late onset diabetes at its earliest stages is critical for efficient and effective intercession and treatment of this debilitating disease. There is a need for a non-invasive diagnostic assay that is reliable at or before the earliest manifestations of late onset diabetes symptoms. There is also a need for developing therapeutic regimens or drugs for treating both the symptoms of diabetes mellitus and of the disease itself.
The present invention satisfies these needs for a useful diagnostic and effective treatment of late onset diabetes and provides related advantages, as well.
The present invention relates to the identification of defects in mitochondrial function, which segregate with late onset diabetes. The invention provides methods for detecting such defects as a diagnostic for late onset diabetes, either before or after the onset of clinical symptoms. More specifically, the present invention provides a method for detecting the presence of or risk of developing diabetes mellitus in a subject comprising comparing (i) a mutational burden at one or more nucleotide positions in an ATP synthase gene in a sample from the subject with (ii) the mutational burden at one or more corresponding nucleotide positions in a control sample, and therefrom identifying the presence of or risk of developing diabetes mellitus. In certain embodiments the mutational burden relates to a mutation in an ATP synthase gene at nucleotide position 8371, 8374, 8383, 8386, 8392, 8395, 8396, 8398, 8401, 8404, 8410, 8419, 8422, 8423, 8428, 8450, 8459, 8463, 8467, 8470, 8473, 8474, 8485, 8486, 8487, 8488, 8491, 8503, 8506, 8508, 8509, 8512, 8539, 8541, 8557, 8562, 8566, 8568 or combinations thereof. In certain other embodiments, at least one mutation is a silent mutation, missense mutation, or combination thereof.
In other embodiments the invention provides a method for detecting the presence of or risk of developing diabetes mellitus in a subject, comprising comparing (i) a mutational burden at one or more nucleotide positions in a tRNALys gene in a sample from the subject with (ii) the mutational burden at one or more corresponding nucleotide positions in a control sample, and therefrom identifying the presence of or risk of developing diabetes mellitus. In certain embodiments the mutational burden relates to a mutation in a tRNALys gene at nucleotide position 8336, 8345, 8348, 8349, 8351 or combinations thereof. In certain further embodiments of any of the above embodiments of the invention, the presence of the mutation is detected by a technique that may be hybridization with oligonucleotide probes, a ligation reaction, a polymerase chain reaction or single nucleotide primer-guided extension assay, or variations thereof.
It is another aspect of the present invention to provide a method of detecting genetic mutations which cause diabetes mellitus or indicate a predisposition to develop diabetes mellitus, comprising determining the sequence of at least one mitochondrial ATP synthase gene from humans known to have diabetes mellitus; comparing the sequence to that of the corresponding wildtype mitochondrial ATP synthase gene; and identifying mutations in the humans which correlate with the presence of diabetes mellitus.
Turning to another aspect, the invention provides an isolated nucleotide sequence which is at least partially complementary to a mitochondrial DNA sequence, wherein the isolated sequence contains at least one mutation in an ATP synthase subunit 8/6 gene which correlates with the presence or risk of diabetes mellitus. In another aspect the invention provides an isolated nucleotide sequence which is at least partially complementary to a mitochondrial DNA sequence, wherein the isolated sequence contains at least one mutation in a tRNALys gene which correlates with the presence or risk of diabetes mellitus. In another aspect the invention provides an isolated nucleotide sequence which is at least partially complementary to a mitochondrial DNA sequence, wherein the isolated sequence contains at least one mutation located between mitochondrial DNA nucleotides 8295 and 8571 which correlates with the presence or risk of diabetes mellitus. In certain embodiments of the above aspects of the invention directed to an isolated nucleotide sequence, the isolated sequence is labeled with a detectable agent.
In yet another aspect, the invention provides a method of inhibiting the transcription or translation of one or more mutant ATP synthase encoding nucleic acids correlated with late onset diabetes mellitus, or the transcription of one or more mutant tRNALys encoding nucleic acids correlated with late onset diabetes mellitus, the method comprising contacting the ATP synthase or tRNALys encoding nucleic acids with antisense sequences specific to the mutant nucleic acids; and allowing hybridization between target mutant ATP synthase or tRNALys encoding nucleic acids and the antisense sequences. In certain embodiments, hybridization is performed under conditions wherein the antisense sequences bind to and inhibit transcription or translation of target mutant ATP synthase 8/6 encoding nucleic acid or inhibit transcription of target mutant tRNALys encoding nucleic acid without preventing transcription or translation of the corresponding wild-type nucleic acids or other mitochondrial genes. In certain embodiments, the mutant ATP synthase or tRNALys encoding nucleic acids are RNA.
The present invention also provides, in another aspect, a method for evaluating a compound for use in diagnosis or treatment of diabetes mellitus, the method comprising contacting a predetermined quantity of the compound with cultured cybrid cells having genomic DNA originating from a xcfx810 cell line and mitochondria originating from tissue of a human having a disorder that is associated with late onset diabetes mellitus; measuring a mitochondrial complex V activity that is affected by the mutant mitochondrial DNA in said cybrid cells; and correlating a change in the mitochondrial complex V activity with effectiveness of the compound. In on embodiment, the xcfx810 cell line is immortal.
Turning to another aspect, the invention provides a method for evaluating a compound for its utility in the diagnosis and treatment of diabetes mellitus, comprising inducing differentiation of cultured undifferentiated cybrid cells having genomic DNA originating from a xcfx810 cell line and mitochondria originating from tissue of a human having a disorder that is associated with late onset diabetes mellitus; contacting a predetermined quantity of the compound with these cybrid cells; measuring a mitochondrial complex V activity that is affected by mutant mitochondrial DNA in the cybrid cells and correlating a change in the mitochondrial complex V activity with effectiveness of the compound in the diagnosis or treatment of diabetes mellitus. In certain embodiments the xcfx810 cell line is immortal.
In another embodiment the invention provides a method for detecting the presence of diabetes mellitus in a human subject, comprising the steps of obtaining a biological sample containing mitochondria from the subject; and determining the presence of at least one polypeptide encoded by a mitochondrial ATP synthase gene. In certain embodiments the presence of the polypeptide is determined with at least one monoclonal antibody or polyclonal antibody.
It is another aspect of the present invention to provide a method for detecting the presence or risk of developing diabetes mellitus in a human, the method comprising determining the presence in a biological sample from a human of a nucleic acid sequence having a mutational burden at one or more nucleotide positions in a sequence region corresponding to a wildtype mitochondrial DNA sequence, wherein the mutational burden correlates with the presence of or risk of developing diabetes mellitus.
Another aspect of the invention provides an isolated nucleotide sequence which is at least partially complementary to a mitochondrial DNA sequence, the isolated sequence having a mutational burden at one or more nucleotide positions in a sequence region corresponding to a wildtype mitochondrial DNA sequence, wherein the mutational burden correlates with the presence of or risk of developing diabetes mellitus.
In still another aspect the invention provides a method for evaluating a compound for use in diagnosis or treatment of diabetes mellitus, comprising contacting a predetermined quantity of the compound with cultured cybrid cells having genomic DNA originating from a xcfx810 cell line and mitochondria originating from tissue of a human having a disorder that is associated with late onset diabetes mellitus; measuring a phenotypic trait in the cybrid cells that correlates with the presence of the mitochondria and that is not present in cultured cybrid cells having genomic DNA originating from a xcfx810 cell line and mitochondria originating from tissue of a human free of a disorder that is associated with late onset diabetes mellitus; and correlating a change in the phenotypic trait with effectiveness of the compound.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth herein which describe in more detail certain aspects of this invention, and are therefore incorporated by reference in their entireties.